Regulated transitions through early B cell differentiation are of great importance for the generation and selection of the mature B cell repertoire. We have recently described a transitional phase in early mouse B cell differentiation that in addition to pre-BCR and IL7R signaling also displays integrin-dependent stromal adhesion, strong proliferative capacity and self-renewal. Loss in the transcriptional regulator Ikaros causes arrest at this stromal- adherent pre-B cell stage with a profound increase in integrin signaling, stromal adhesion and self-renewal and loss in pre-BCR differentiation signaling. Although this stage of early B cell differentiation is also important for the developmen of human hematological disorders, including immunodeficiencies and leukemias, little is known of this process in the human hematopoietic system and is the focus of our current investigation. In the first aim, we will evaluate the hierarchy of human B cell precursors that arise in a bone marrow stromal-dependent system. We will examine the in vitro stromal dependence of early human B cell precursors for proliferative expansion and differentiation and identify the signaling pathways and transcriptional factors associated with these events. The differentiation properties of human B cell precursors generated in vivo in immune-deficient NSG mice will be evaluated in a similar fashion. In the second aim, we will examine the role of IKAROS in human pre-B cell differentiation. The IKZF1 locus will be disrupted by genome editing in human B cell precursors to model mutations in human disease. IKAROS mutant B cell precursors will be tested for differentiation, proliferation and adhesion properties in in vitro stromal cultures and in vivo aftr transplantation in NSG mice. Cellular and molecular data generated from IKAROS deficient human pre-B cell precursors and its normal counterparts can serve as baselines to which hematological disorders can be compared. Together these studies will generate critical new knowledge on the molecular pathways that drive the human developmental process and its aberrant manifestations. This work will enable us to move seamlessly between the mouse and human to discover better approaches for treating hematological disorders.